Volume 26, Issue 2, Pages 361-374.e4 (August 2017) Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake  Carlos Manlio Díaz-García, Rebecca Mongeon, Carolina Lahmann, Dorothy Koveal, Hannah Zucker, Gary Yellen  Cell Metabolism  Volume 26, Issue 2, Pages 361-374.e4 (August 2017) DOI: 10.1016/j.cmet.2017.06.021 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 NADHCYT Transients with Synaptic Stimulation (A) Filmstrip of metabolic transients in dentate granule neurons. A sequence of acquisitions shows the brief increase in RCaMP lifetime reflecting an augmented Ca2+ concentration after synaptic stimulation. The peak in the Peredox lifetime elevation occurs approximately 1 min later, reflecting changes in the cytosolic NADH/NAD+ ratio (NADHCYT) toward a more reduced state. Images are pseudocolored according to sensor lifetimes. The color scale bar to the right shows the range of lifetime values for both RCaMP and Peredox sensors. For the latter, the color scale is accompanied by an axis converting lifetimes to calibrated NADH/NAD+ ratios. (B) Individual traces from a cell in the filmstrip illustrate the time course of synaptically stimulated transients in Peredox (top) and RCaMP1h lifetimes (bottom). (C) Average transient increases in Peredox lifetime after synaptic stimulation of 60 brief pulses delivered at 20 Hz. Traces represent the mean ± SEM (events = 447, neurons = 166, slices = 27, mice = 14; SEM calculated using number of neurons). (D) The magnitude of the NADHCYT increase correlates with the amplitude of the neuronal Ca2+ spike in a wide range of Ca2+ responses elicited by synaptic stimulation. The scatterplot summarizes data resulting from events elicited with different stimulation strengths (events = 554, neurons = 166, slices = 27, mice = 14). (E) Glycolysis and lactate utilization enhance neuronal NADH production. The cartoon summarizes the main pathways leading to an increased cytosolic NADH/NAD+ ratio in neurons. After passive uptake via GLUT transporters, glucose is phosphorylated and enters glycolysis, where NADH is produced by the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lactate import occurs through monocarboxylate transporters (MCT) and is then converted to pyruvate by the enzyme lactate dehydrogenase (LDH), also reducing NAD+ to NADH. Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Glutamate Release Is Neither Sufficient nor Necessary for NADHCYT Transients (A) Typical electrode placement for synaptic stimulation. The picture shows an infrared differential interference contrast image of a horizontal slice from the hippocampus, where the stimulating electrode (s.e.) is placed in the molecular layer (m.l.), and an extracellular recording electrode (r.e.) is inserted in the layer of dentate granule neurons (DGNs, following the arrow direction). The square outline shows the area used for biosensor imaging. (B) Inhibition of ionotropic glutamate receptors abolishes the metabolic transients. A representative trace of a synaptically stimulated DGN (using a train of 60 pulses at 20 Hz) shows that the Peredox signal disappears after blocking synaptic transmission with a mixture of 5 μM NBQX and 25 μM D-AP5 in the artificial cerebrospinal fluid (ACSF). The complete blockade of synaptic transmission is corroborated by the absence of the typical RCaMP1h signal after stimulation. The effect of synaptic blockers was partially reversed after 5 min of washout in regular ACSF, and both Ca2+ and NADH transients were completely recovered after 10 min in ACSF. (C) Dataset for the effect of ionotropic glutamate receptor block. Data were compared by a paired Student's t test (neurons = 46, slices = 6, mice = 3). (D) Typical electrode placement for antidromic (axonal) stimulation. In this experimental paradigm, the stimulating electrode (s.e.) is placed in the hilus (hil) and an extracellular recording electrode (r.e.) is inserted in the layer of DGNs, following the arrow direction. The square outline shows the area used for biosensor imaging. (E) Average transient increases in Peredox lifetime after antidromic stimulation of 100 brief pulses delivered at 50 Hz. Traces represent the mean ± SEM (events = 179, neurons = 84, slices = 18, mice = 11; SEM calculated using number of neurons). The lower panel shows the putative average Ca2+ spike in the cells, as a proxy for neuronal excitation. (F) Elevation in the NADH/NAD+ ratio is directly correlated with the neuronal Ca2+ increase after electrical antidromic stimulation in DGNs, similarly to synaptic stimulation. Scatterplots of changes in Peredox lifetime versus variations in RCaMP lifetime for events recorded after antidromic stimulation (open circles; events = 205, neurons = 87, slices = 19, mice = 12), overlap with data collected using synaptic stimulation (dark squares). Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Neuronal Lactate Import Is Not Required for the NADHCYT Transients (A) Monocarboxylate transporter (MCT) inhibition abolishes NADH transients induced by the application of external lactate, but enhances the NADHCYT increase after synaptic stimulation. The MCT inhibitor AR-C155858 (2 μM, 5 min) prevents the increase in the NADH/NAD+ ratio in response to external lactate (10 mM). However, the endogenous NADH transients in response to synaptic stimulation (60 pulses at 20 Hz) were bigger in the presence of the MCT inhibitor (MCT inhib). (B) Lactate-induced changes in Peredox lifetime were significantly reduced in the presence of the MCT inhibitor. Data were compared by a paired Student's t test (neurons = 36, slices = 5, mice = 5). (C) Scatterplots of changes in Peredox lifetime versus changes in RCaMP lifetime, show that, as in (A), acute (5 min) or long-lasting (>10 min) exposure to the MCT inhibitor increases the size of NADHCYT transients for similar Ca2+ transients, making the slope of the positive correlation steeper. All comparisons were made using a paired Student's t test. Control data points are open symbols, while symbols for treatments are filled, using circles or squares for acute (neurons = 39, slices = 7, mice = 5) and long-lasting (neurons = 36, slices = 5, mice = 5) application of the MCT inhibitor respectively. The dashed lines and surrounding shaded zones show the linear fit and 95% confidence interval for the (unpaired) data with (black) and without (gray) MCT inhibitor; the lower dashed line shows the prediction of the ANLS that the stimulation-induced transients should be reduced comparably with the lactate-induced transients in (B). (D) Lactate dehydrogenase (LDH) inhibition also decreases the NADHCYT transients induced by the application of external lactate and increases the NADH/NAD+ ratio after synaptic stimulation. (E) The LDH inhibitor GSK-2837808A (2 μM, 30 min) diminishes the change of the cytosolic NADH/NAD+ ratio in response to external lactate (10 mM). Data were compared by a Wilcoxon signed-rank test (neurons = 54, slices = 5, mice = 5). (F) Scatterplots of changes in Peredox lifetime versus changes in RCaMP lifetime, show that, as in (A), sustained (>30 min) exposure to the LDH inhibitor increases the size of NADHCYT transients for similar Ca2+ transients, making the slope of the positive correlation steeper. Control data points are open circles (events = 179, neurons = 54, slices = 5, mice = 5), while black squares correspond to LDH inhibition (events = 54, neurons = 54, slices = 5, mice = 5). The dashed lines and surrounding shaded zones show the linear fit and 95% confidence interval for the (unpaired) data with (black) and without (gray) LDH inhibitor; the lower dashed line shows the prediction of the ANLS that the stimulation-induced transients should be reduced comparably with the lactate-induced transients in (E). (G) Left: representative traces of changes in CFP/YFP fluorescence emission ratio for Laconic before and after the application of the MCT inhibitor (2 μM, 5 min). Right: the relative increase in the CFP/YFP ratio was unaffected by MCT inhibition (neurons = 41, slices = 5, mice = 4; Wilcoxon signed-rank test, p > 0.05). Box plots indicate the middle 50% of the data, and whiskers show the 5%–95% range; shaded zones show the 95% confidence interval of the median. Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 NADHCYT Transients Most Likely Arise from Neuronal Glycolysis (A) Treatment with the GAPDH inhibitor iodoacetic acid (IAA) reduces the NADHCYT transients in response to antidromic stimulation. Representative trace of NADHCYT transients elicited by antidromic stimulation (same conditions as Figure 2), before and after IAA treatment. The bars indicate the times of application of both lactate (2 mM) and IAA (0.5 mM). IAA inhibits glycolysis irreversibly; the temperature was lowered (shaded zone) before and during IAA application to minimize damage to the slice's health. (Temperature affects sensor lifetime directly, which is why there are large changes in the sensors' response during the temperature changes.) After this treatment, the cells were stimulated again, leading to a much smaller NADHCYT transient than before IAA treatment. (B) IAA decreased the ability of neurons to produce NADH in response to excitation, as observed in the reduction of the Δ(Peredox LT)/Δ(RCaMP LT) ratio (neurons = 18, slices = 5, mice = 5). This ratio normalizes the NADHCYT change to the size of the Ca2+ transients, as they are correlated (Figures 1 and 2), and the Ca2+ transients are somewhat affected by IAA treatment. Figure S4 shows additional controls and analysis. Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Stimulation Induces Transient Dips of Neuronal Intracellular Glucose, Consistent with Increased Neuronal Glycolysis (A) Representative trace of glucose dips elicited by synaptic stimulation of 60 pulses at 50 Hz. Glucose transporter inhibition by 0.2 μM cytochalasin B (CytoB, 10 min) increased the size of the glucose dips. Figure S5 shows the properties of the SweetieTS sensor. (B) The structural analog cytochalasin D (CytoD, 0.2 μM, 10 min) does not affect the glucose dips. This experiment serves as a negative control for the possible effects of CytoB on the cytoskeleton. The effect sizes for CytoB (neurons = 22, slices = 4, mice = 4) and CytoD (neurons = 17, slices = 4, mice = 3) were compared using an unpaired Student's t test. (C) Representative recording of glucose dips in the continuous presence of 0.2 μM CytoB (to enhance the glucose dips), before and after the application of MCT inhibitor (2 μM). (D) The acute application of 2 μM MCT inhibitor (5 min) reduced the glucose dips. Comparisons were done using the ratio between two consecutive control pulses and the ratio between the dip in the presence of MCT inhibitor and the previous control pulse (neurons = 28, slices = 7, mice = 4). The nonparametric Wilcoxon signed-rank test was used because at least one of the distributions was not Gaussian. Box plots indicate the middle 50% of the data, and whiskers show the full range; shaded zones show the 95% confidence interval of the median. Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 NADHCYT Transients Occur Independently of the Mitochondrial Malate-Aspartate Shuttle (A) Inhibition of the malate-aspartate shuttle (MAS) by aminooxyacetate (AOA) increases the cytosolic NADH/NAD+ ratio. The right panel shows representative traces of Peredox lifetime in two neurons, which increases after the application of 10 mM AOA in the ACSF. In the control cell, Peredox LT remains high in the continuous presence of 10 mM AOA, whereas the exposure of the second cell to 1.5 mM pyruvate (Pyr) decreased the Peredox LT to baseline levels, even in the presence of the shuttle inhibitor. The right panel summarizes the effect of AOA and pyruvate (as well as their combination) on Peredox lifetime (neurons = 40, slices = 5, mice = 5). Values are significantly different from each other with a p < 0.01, except for the conditions 1.5 mM Pyr and 1.5 mM Pyr + 10 mM AOA. (B) Representative traces of activity-induced changes in Peredox lifetime of a neuron before and after AOA application, both obtained in the presence of 1.5 mM pyruvate in the ACSF. Antidromic stimulation was delivered in the presence of picrotoxin (100 μM) in addition to the ionotropic glutamate receptor blockers. This demonstrates that neuronal activation still produces an increase in the cytosolic NADH/NAD+ ratio, despite MAS inhibition. (C) The size of the NADHCYT transient is marginally affected by MAS blockade. Left: the repetitive exposure to pyruvate increases the NADHCYT transients around 11% in slices that were not treated with AOA (neurons = 33, slices = 3, mice = 3). By contrast, the transients obtained in the simultaneous presence of AOA and pyruvate are ∼8% smaller than the initial transients in the presence of pyruvate alone (neurons = 40, slices = 5, mice = 5; Wilcoxon matched-pairs signed-rank test, ∗p < 0.05, ∗∗p < 0.01). Right: for comparisons, we took into account the hysteresis effect by dividing the Δ(Peredox LT)/Δ(RCaMP LT) ratio at the final condition (second exposure to pyruvate in controls or AOA plus pyruvate in treated neurons) by the initial pulse in the presence of pyruvate alone. NADHCYT transients in the presence of AOA plus pyruvate, are approximately 19% smaller than expected (Mann-Whitney test, ∗∗p < 0.01). Box plots indicate the middle 50% of the data, and whiskers show the 5%–95% range; shaded zones show the 95% confidence interval of the median. Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 NADHCYT Transients In Vivo with Sensory Stimulation (A) Representative pseudocolored lifetime image of neurons expressing RCaMP1h (left panels) and nuclear-localized Peredox (right panels) in layers II/III of the primary somatosensory cortex of an awake mouse. Scale bar, 10 μm. The images were acquired 0.33 min before (top panels), during (middle panels), and 1 min after (bottom panels) whisker stimulation. Stimulation resulted in a brief increase in RCaMP lifetime in the responsive neuron, reflecting an augmented Ca2+ concentration (middle left panel). This was followed approximately 1 min later by an increase in Peredox lifetime for the responsive neuron, reflecting changes in the cytosolic NADH/NAD+ ratio (NADHCYT) toward a more reduced state (bottom right panel). The color scale bar to the right shows the range of lifetime values for both RCaMP1h and Peredox sensors. (B) Average transient increases, within neuronal regions of interest, in cytosolic NADH/NAD+ ratio (top panel) and Ca2+ concentration (bottom panel) following whisker stimulation (red arrow) consisting of 10 s of mechanical stimulation at 5 Hz. Traces represent the mean ± SEM; n = 19 neurons, N = 9 mice (3 males, 6 females). (C) Elevation in the NADH/NAD+ ratio is directly correlated with the increase in intracellular Ca2+ following whisker stimulation. (D) Comparison of lactate-induced Δ(Peredox LT) (50 mM lactate) before and after MCT inhibition (50 μM AR-C155858) in vivo using a paired Student's t test. n = 59 neurons, N = 6 mice (3 females, 3 males). (E) Representative experiment on the effect of MCT inhibition on neuronal NADHCYT transients elicited by whisker stimulation. In vivo application of MCT inhibitor AR-C155858 (50 μM, 20 min) did not reduce the endogenous NADH transients in response to whisker stimulation (10 s of mechanical stimulation at 5 Hz). (F) The scatterplot of Δ(Peredox LT)/Δ(RCaMP LT) shows that in vivo exposure to MCT inhibitor does not reduce the size of the NADH/NAD+ transients and the slope of the relationship. The ratio of stimulation-induced Δ(Peredox LT)/Δ(RCaMP LT) in the absence of MCT inhibitor was 0.13 with a 95% confidence interval of 0.10–0.16. In the presence of MCT inhibitor, the ratio of stimulation-induced Δ(Peredox LT)/Δ(RCaMP LT) was 0.15 with a 95% confidence interval of 0.11–0.20. n = 4 neurons, N = 3 mice (2 females, 1 male). The dashed line shows the prediction of the ANLS that the stimulation-induced transients should be reduced comparably with the lactate-induced transients in (D) (based on adjusting the slope for the linear fit of the control measurements). Cell Metabolism 2017 26, 361-374.e4DOI: (10.1016/j.cmet.2017.06.021) Copyright © 2017 Elsevier Inc. Terms and Conditions